A considerable number of DNBA restriction fragment length polymorphisms (RFLPs) have been discovered and genotyped in populations and in reference families. RFPL's result from single base-pair differences within restriction enzyme recognition sequences which lead to fragments of variable length. Considerable population data has been assembled by "Southern analysis', a laborious procedure requiring micrograms of DNA and hybridization with labeled probes. We have devised an innovative methodology for rapid conversion of RFPLs into a PCR-based format allowing reliable, cost-effective genotyping of individuals on a large scale. We will develop RFPL genotyping kits for approximately 200loci distributed throughout the human genome as well as kits for rapid sequencing of any RFPL. Phase I includes: location and sequencing of approximately 500bp DNA domains containing variable restriction sites in plasmid, phage and cosmid clones, conversion from Southern to PCR analysis, development of a prototype kit for genotyping 3 RFPLs in a multiplex assay and applying it to 1 individual from each of 19 populations, and international field testing. The genotyping kits are intended for 4 major users: (1) laboratories in the Human Genome Diversity project, (2) geneticists performing linkage analysis of complex disorders, (3) epidemiologists pursuing disease association studies with molecular markers and (4) forensic investigators solving identification and paternity cases.